The present invention relates to recombinant lactoferrins (rLf), their production from plants, and uses thereof.
The use of certain proteins found in mammals has been questioned due to the possibility of contamination by non conventional infectious agents, particularly of the prion type. The impact on marketing and regulations is great. The development of proteins free of all animal contamination is, therefore, a new possibility but one which faces new difficulties depending on the protein and the plant matter chosen.
Prior art includes the publication by Mitra et al. (1994), which describes the transformation of tobacco cells with the DNA sequence which codes for human lactoferrin. However, this publication is limited to plant cells, and does not allow for the production of regenerated plants from these cells. Furthermore, the protein expressed is not purified, and seems, in any case, imperfect as only a part (48 kDa) is detected, and not the entire protein.
In the prior art, application WO 9637094 is also known; this describes the production of plants which are resistant to viruses, by means of transforming them with a gene which codes for lactoferrin. However, in this document as well, the protein is not purified and the only indication of its presence is a Weston blot test.
As a result of the complexity of lactoferrin and the characteristics of the plant matter used, the extraction and purification procedures also represent major obstacles in the production of lactoferrin from plants. The difficulties posed by these two procedures can represent a new set of problems for each protein which one wishes to produce from a plant.
Lactoferrin is a glycoprotein of the transferrin family. Following its discovery in human milk, lactoferrin was shown to be present in many other species such as cows, goats, pigs, mice and guinea pigs, but a highly variable concentrations. In human milk, the concentration of lactoferrin is in the range of 1 to 2 g/l; the concentration is particularly high in colostrum and diminishes over the course of lactation. In milk, lactoferrin is present primarily in the apo form, that is to say, unsaturated in iron. Lactoferrin has also been found in many other secretions, such as the saliva, bile, pancreatic fluid, and secretions of the small intestine. It is found in most mucus, such as bronchial, vaginal, nasal and intestinal secretions.
Lactoferrin is also present in polymorphonuclear neutrophilic leucocytes, where it is localized in the secondary granules of cells that do not contain myeloperoxidase. Leucocytic lactoferrin is synthesized during granulopoiesis of the promyelocyte stage of the metamyelocyte stage. When the neutrophils degranulate, lactoferrin is released into the plasma at a relatively low concentration (0.4 to 2 mg/l) as compared with the level of transferrin found in the blood (2 to 3 g/l).
The peptide sequence for human lactoferrin (hLf) was determined in 1984 by Metz-Boutigue et al. This sequence of 692 amino acids was confirmed by way of cloning of the cDNA for lactoferrin of the human mammary gland (Powell and Ogden, 1990, Rey et al., 1990). Lactoferrin and serotransferrin have very similar primary structures and spatial configurations. Their polypeptide chains are formed of two lobes (N terminal lobe and C terminal lobe) joined by a small alpha helix peptide. Sequence similarities between the N and C terminal halves of human lactoferrin reach 37%. Trypsic hydrolysis of human lactoferrin allowed Legrand et al. (1984) to produce the 30 kDa N trypsic (N-t) fragment (residues of amino acids 4 to 283), and the 50 kDa C trypsic (C-t) fragment (residues of amino acids 284 to 692). At equimolar proportions, these fragments can reunite to form a non-covalent N-t/C-t complex which has electrophoretic and spectroscopic characteristics similar to those of human lactoferrin (Legrand et al, 1986).
Lactoferrin can reversibly bind two ferric ions which results in a salmon-pink coloration, the maximum absorption of which is centered at 465 nm. The binding of each ferric ion requires the same of a carbonate ion. At a pH of 6.4, the association constant of the complex [Fe3+]2-Lf is in the range of 1024 Mxe2x88x921, which decreases with pH. X-ray diffraction study of human lactoferrin at 3.2 xc3x85 to 2.8 xc3x85 and at 2.2 xc3x85 show that each ferrous ion is coordinated with 2 tyrosine residues, a histidine, an aspartic acid and a carbonate ion. These amino acids are the same in both lobes. Both lactoferrin iron binding sites have a strong affinity for this metal, but they release it at different pH levels. The N-t lobe releases its iron at pH 5.8 (acid labile lobe), while the C-t lobe (acid stable lobe) releases its iron at pH 4. Other ions may bind to the protein, in particular gallium. Researchers interested in the use of a 67Ga-Lf complex as a tracer in cancer diagnostics have shown that following injection with 67Ga, the complex is preferentially found in the mammary tissues, in physiological and pathological secretions and in Burkitt and Hodgkin lymphomas.
In terms of glycosylation, lactoferrin isolated from human milk has three glycosylation sites, Asn138, ASN479 AND Asn624, the first located on the N-t lobe and the other two on the C-t lobe. Glycosylation occurs preferentially at two sites (Asn138 and ASN479) in 85% of molecules, while glycosylation of one site (Asn479) and of the three sites simultaneously happens in 5% and 9% of cases, respectively. Lactoferrin glycans are of the mono or disialylated and fucosylated N-acetyllactosamine type (Spik et al, 1982). The fucose residues are a (1,6) branched on the N-acetylglucosamine 5xe2x80x2 of the attachment point, or are xcex1 (1,3) branched on the N-acetylglucosamine 5xe2x80x2 of the antenna. Leucocytic lactoferrin differs from that above by the total absence of fucose.
While serotransferrin is unquestionably the principal transporter of iron in all the cells of the organism, the roles played by lactoferrin appear to be essentially linked to defense of the organism and inflammatory mechanisms, working either directly on pathogenic micro-organisms, or indirectly on the effector immune cells.
Lactoferrin is an anti-microbial agent which works by way of several mechanisms. The first of these is the bacteriostatic effect of lactoferrin by means of iron deprivation (Spik et al., 1978). By taking up iron from its surroundings, lactoferrin inhibits bacteria division, as iron is an indispensable element in the biosynthesis of DNA. Furthermore, a more complex mechanism which causes antibodies to act has been shown. The bacteriostatic activity of lactoferrin increases in the presence of the specific IgA and IgG of the pathogenic bacterium. At the same time, lysozyme can associate its lytic activity on the walls of Gram+ bacteria with the action of lactoferrin. Thus, in milk, lactoferrin, lysozyme and antibodies can act synergistically in case of microbe attack.
The second anti-bacterial effect of lactoferrin is linked to it bactericidal capacity. Lactoferrin appears to bind to the walls of Gramxe2x88x92 bacteria, which appears to destabilize them and to provoke the release of lipopolysaccharides (LPS). Thus it would seem that the walls become more fragile and more susceptible to the effects of hydrophobic antibiotics. These theories have been confirmed by use of electron microscopy which shows the destabilizing effects of lactoferrin on Gramxe2x88x92 bacteria, including E. coli. A hypothesis has been made to the effect that the binding of lactoferrin occurs on the A lipid of the LPS, and that this is followed by the extraction of these LPS from the external membranes of the bacteria, irreparably damaging them. The bactericide regions of human lactoferrin (lactoferrin A) and bovine lactoferrin (lactoferrin B) have recently been identified. They are both found in an N-terminal lobe loop comprising 18 amino acids. This loop is formed by a disulphur bridge between the residues Cys 30 and 37 for human lactoferrin, and 19 and 36 for bovine lactoferrin. In addition, the importance of the loop formed by residues 28-34 of human lactoferrin in its binding to LPS has been demonstrated.
Thus, due to its bacteriostatic and bactericide activities, lactoferrin present in human milk protects nursing infants from infantile diarrheas.
An anti-fungal effect has been established for lactoferrin with respect to several strains of Candida. Studies have also been carried out with bovine lactoferrin showing this action both on yeasts and on filamentous fungi. Also, the effect of bovine lactoferricin appears to be greater than that of whole bovine apolactoferrin and similar to that of polymyxin B, an antibiotic of the cationic peptide type, known for its membrane destabilisation properties. It has also been shown that lactoferrin B interacts directly with the surface of the fungus, thereby introducing changes to its ultrastructure.
Recently, an antiviral activity has been demonstrated for lactoferrin by several authors. Certain types of virus penetrate the cells by means of a mechanism which causes absorption of proteoglycans by the membranes of the target cells, followed by binding at a specific receptor and fusion of the viral membrane with that of the host. As it is highly basic, lactoferrin binds to the heparan sulphates of cells, and is thereby able to inhibit the adsorption of several types of virus. Highly conclusive in vitro studies have been carried out using HIV (human immunodeficiency virus) and HCMV (human cytomegalovirus) with a CI50 in the range of 10 xcexcg/ml. Similar results have been achieved with HSV-1 (herpes simplex virus type 1). This work showed not only blocking of virus receptors (heparan sulphates, proteoglycans, LDL receptor), but also a possible interaction between lactoferrin and the virus. These recently discovered roles represent new possibilities for prevention and treatment, particularly in terms of immunodeficiency illnesses or recurrent illnesses, in terms of these viral infections.
One of the consequences of tissue inflammation is the formation of free radicals and the peroxidation of lipids. The formation of these free radicals results particularly from the phagocytosis mechanisms of the micro-organisms. According to the Haber-Weiss reaction, free radicals are created as the result of ferric iron, which acts as a reaction catalyst. It has been shown in vivo that lactoferrin, coming from the degranulation of neutrophils, stops the formation of extra-cellular free radicals by immediately taking up the iron which would catalyse the formation of these radicals. As the result of this action, lactoferrin prevents tissues from being damaged. By means of a similar mechanism, lactoferrin inhibits the peroxidation of lipids and thus protects the cellular membranes to which it binds. These studies also showed that lactoferrin has anti-oxidising and anti-inflammatory effects if the protein is in the form without iron.
Lactoferrin released by leucocyte granules has been identified as an inhibitor of granulocyte and macrophage colonies by reducing the production of GM-CSF (granulocyte and macrophage colony stimulation factor). The mechanism involved seems complex, as lactoferrin appears to act by inhibiting the release of a monokine, which is itself responsible for the release of GM-CSF by the lymphocytes, the fibroblasts and the endothelial cells. This monokine has been identified at IL1.
Many of the roles played by lactoferrin, such as the suppression of the production of antibodies, regulation of complement activation and regulation of NK (natural killer) cells activity, suggest mechanisms which are regulated by cytokines. Studies show that lactoferrin can exert a negative retrocontrol on certain cytokines such as the IL1, the IL2 and the TNFa, so as to prevent the activation of leucocytes in inflammation areas.
Zimecki et al (1991) showed that immature CD4xe2x88x92 CD8xe2x88x92 thymocytes incubated in the presence of lactoferrin acquire the marker CD4+ characteristic of auxiliary lymphocytes. These authors also point out various phenotypic and functional changes in B cells in the presence of lactoferrin (Zimecki et al., 1995). A specific receptor for human lactoferrin has been defined for activated lymphocytes (Mazurier et al., 1989). In addition, the site of interaction between lactoferrin and its lymphocytic receptor has been described: Legrand et al. (1992) showed that it was contained in the N terminal region, and more specifically, in the first 50 residues of lactoferrin.
Lactoferrin appears to regulate the cytotoxic activity of NK (natural killer) cells and LAK cells at very low quantities (0.75 xcexcg/ml). Lactoferrin significantly increases the cytotoxic activity of NK cells with respect to tumour cells and cells infected by retroviruses. Lactoferrin also effects, the cytotoxicity of monocytes. The cause of this stimulation by lactoferrin of the cytotoxic activity of NK cells, of lymphocytes and also adherent cells may be the result of either activation of the killer cells following the internalisation of lactoferrin, or of a modification of the target cells which then become more susceptible to lysis.
Lactoferrin also seems to play a immunoregulatory role in inflammatory responses (Elass-Rochard et al., 1995; confirmed by Elass-Rochard et al., 1998). Demonstration of anti-tumour activity was also the object of studies by Salmon et al. (1998).
Lactoferrin has a growth factor effect on various cells in environments lacking in foetal veal serum. This activity has been demonstrated, particularly on the B and T lymphocytic strain with respect to a macrophage murine strain (P388 DI). Lactoferrin also stimulates the incorporation of thymidine tritiate in the DNA of rat enterocytic cells.
The role of iron in growth factor activity is still an object of controversy. According to some authors, lactoferrin works by providing the iron necessary to cellular proliferation. Others hold that iron is not involved, and that the mitogen activity is due solely to the protein itself.
The hypothesis to the effect that lactoferrin is involved in the intestinal absorption of iron results from the observation that amongst breast-fed infants, the incidence of iron deficiency is very low. In fact, only such children maintain a major supply of iron up to the age of 6 months, which implies high bioavailability for iron contained in human milk. The percentage of absorption can reach 81% during the first three months of life, and diminishes rapidly thereafter. Amongst the various components of human milk, lactoferrin is the best candidate for explanation of both the great bioavailability of iron in milk and regulation of its absorption. Cox et al. studied the importance of lactoferrin in the absorption of iron by the human intestine as early as 1979. The enterocytic receptor for lactoferrin was first shown to be present in rabbits, then in mice. Finally, the human enterocytic receptor was studied. It was shown that, with HT29 enterocytic cell cultures, the number of lactoferrin receptors doubled in the presence of iron chelator (Mikogami et al., 1994, 1995). This increase is due to a de novo synthesis of receptors. The expression of the lactoferrin receptor is, therefore, regulated by a lack of iron, and this lack also induces an increase in the internalisation of the lactoferrin receptor by the enterocytes. Thus, lactoferrin seems to be involved in iron nutrition, in particular, in case of iron deficiencies.
Not only has the cDNA sequence for human lactoferrin been determined, but the cDNA sequence for various animal species has been established. Specifically, sequences have been found for:
bovine lactoferrin (Pierce et al., 1991)
porcine lactoferrin (Alexander B. F. et al., 1992)
murine lactoferrin (Shirsat N. V. et al., Gene, 1992)
caprine lactoferrin (Le Provost F. et al., 1994).
Analysis of peptide sequences corresponding to cDNA sequences indicates major similarities. In particular, the human lactoferrin sequence shows a 69% correspondence with that of cows. As with human lactoferrin, the nucleotide sequence of bovine lactoferrin cDNA contains a signal peptide composed of 16 amino acids.
This structural similarity results in functional similarities. In particular, as the N-terminal end of both bovine and human lactoferrin have a great number of basic amino acids, these two lactoferrins are recognised in a similar way by many constituent acids. It should be noted, for example, that these two proteins have similar interactions with the lipopolysaccharides of Gramxe2x88x92 bacteria and the proteoglycans which are found on the surface of many cells. (Elass-Rochard E., et al., 1995).
These interactions appear to imply that these two lactoferrins may play the same role in the inhibition of release of cytokines of macrophages activated by lipopolysaccharides, and therefore in inflammatory reaction mechanisms.
In the same manner, these two lactoferrins show the same binding characteristics for enterocytic cells and, therefore, play the same role in intestinal iron absorption and various anti-bacterial and anti-viral activities.
Thus, the many roles played by lactoferrin, and especially its antioxidant, anti-microbial and anti-viral activities make this protein of major importance in terms of treatment, and above all prevention, of bacterial, fungal and viral infections, and also in terms of the prevention of septic shock which, classically, occurs after surgical operations.
The immunoregulatory and anti-tumour activities of lactoferrin can also be used in the treatment of inflammation and cancer (Denis et al.; Zimecki et al.).
Lactoferrin can also be used in dermo-pharmaceutical and cosmetic treatments, for example in a cosmetic anti-free-radical treatment, or to protect hair keratin against atmospheric damage.
The invention concerns:
the use of a recombinant nucleotide sequence containing both: a cDNA coding for a lactoferrin, particularly for mammalian lactoferrin, preferably bovine, porcine, caprine or human lactoferrin, or derived proteins (derived protein meaning any protein having at least 70% correspondence with the target protein, particularly at least 80%, for example between 85 and 100% correspondence and/or having a different glycosylation profile while maintaining the functional characteristics of the reference lactoferrin); and the elements required by a plant cell to produce the lactoferrin or derived proteins coded by said cDNA, particularly a transcription promoter and terminator recognised by the transcriptional machinery of plant cells, so as to produce from these cells, or from plants produced therefrom, lactoferrin or derived proteins.
a recombinant nucleotide sequence characterised in that it contains both: a sequence coding for lactoferrin, particularly for mammalian lactoferrin, preferably bovine, porcine, caprine or, human lactoferrin, or derived proteins; and the elements required by a plant cell to produce the lactoferrin or derived proteins coded by said cDNA, particularly a transcription promoter and terminator recognised by the transcriptional machinery of plant cells. Advantageously, the nucleotide sequence contains a sequence coding for a signal peptide responsible for the secretion of recombinant polypeptides.
a vector, particularly a plasmid, containing a nucleotide sequence according to the invention inserted, as necessary, at a site which is not essential for its replication.
a cellular host, particularly any bacteria such as Agrobacterium tumefaciens transformed by a vector according to the invention,
a method of producing lactoferrin, particularly human lactoferrin, or derived proteins, characterised in that it comprises:
transformation of plant cells, particularly by means of a cellular host according to the invention, this itself having been transformed by a vector according to the invention, so as to integrate a recombinant sequence according to the invention within the genome of these cells,
as necessary, the production of transformed plants from the aforementioned transformed cells,
recuperation of recombinant lactoferrin, particularly human lactoferrin, or derived proteins produced in said aforementioned transformed cells or plants, particularly by extraction followed, as necessary, by purification,
a genetically transformed plant, plant extract or part of a plant, particularly leaves and/or fruits and/or seeds and/or plant cells, characterised in that it contains one (or several) recombinant nucleotide sequence(s) according to the invention which is (are) integrated in a stable manner in the genome thereof, these plants being particularly chosen from amongst rape, tobacco, maize, peas, tomatoes, carrots, wheat, barley, potatoes, soy, sunflower, lettuce, rice, alfalfa and beets.
a lactoferrin, particularly a human lactoferrin, or a derived protein, characterised in that it is obtained by means of the method of the invention,
a genetically transformed plant, plant extract or part of a plant, particularly leaves and/or fruits and/or seeds and/or plant cells, characterised in that it contains a lactoferrin, particularly a human lactoferrin or a derived protein, according to the invention, these plants being particularly chosen from amongst rape, tobacco, maize, peas, tomatoes, carrots, wheat, barley, potatoes, soy, sunflower, lettuce, rice, alfalfa and beets.
the use of plants, plant extracts or parts of plants and/or proteins (lactofrrrin, particularly human lactoferrin, or derived proteins) according to the invention, for the production of pharmaceutical, medical, odontological, cosmetic or biotechnological compositions,
a biomaterial or a pharmaceutical, medical, odontological, cosmetic or biotechnological composition characterised in that this comprises plants, plant extracts or parts of plants according to the invention, and/or proteins (lactoferrin, particularly human lactoferrin, or derived proteins) according to the invention.
A pharmaceutical composition according to the invention includes particularly any composition according to the invention which constitutes or is used in the manufacture of a composition allowing for the detection or treatment of a pathology or a symptom of bacterial, fungal or viral origin, an inflammation or a pathology having an inflammatory component, septic shocks, a pathology related to a cellular growth phenomenon or an iron deficit, such as anaemia.
A cosmetic composition according to the invention includes particularly any composition according to the invention constituting (or used in the manufacture of) an additive for preparations (creams, ointments, makeup, salves).
Advantageously, the recombinant sequences according to the invention contain one (or several) sequence(s) which code for a peptide responsible for addressing recombinant polypeptides in a specific compartment of the plant cell, particularly in the endoplasmic reticulum or in the vacuoles, or even outside the cell in the pectocellulosic wall or in the extracellular space know as the apoplasm.
Amongst the transcription terminators which can be used for the transformation of plant cells within the framework of the present invention, examples include terminator polyA 35S of the cauliflower mosaic virus (CaMV), or the terminator polyA NOS, which corresponds to the non-coding 3xe2x80x2 region of the nopaline synthase gene of the plasmid TI of the nopaline strain of Agrobacterium tumefaciens. 
Accordingly, the invention takes as its object any recombinant nucleotide sequence such as described above which contains the terminator polyA 35S of the CaMV, or the terminator polyA NOS of Agrobacterium tumefaciens downstream of said cDNA or a derived sequence thereof.
Amongst the transcription promoters which can be used for the transformation of plant cells in the framework of the invention, examples include:
the promoter 35S (P35S), or advantageously the double constitutive promoter (Pd35S) of the CaMV: these promoters allow for expression of the recombinant polypeptides of the invention in the entire plant produced from transformed cells according to the invention, and are described in the article of Kay et al., 1987,
the promoter PCRU of the radish cruciferin gene allows for the expression of the recombinant polypeptides of the invention in only the seeds (or grains) of the plant produced from transformed cells according to the invention and is described in the article by Depigny-This et al., 1992;
the promoters PGA1 and PGA6, which correspond to the non-coding 5xe2x80x2 region of the seed reserve protein genes, GEA1 and GEA6 respectively, of Arabidopsis thaliana (Geubier et al., 1993) allow for specific expression in seeds,
the chimeric promoter super-promoter PSP (Ni M. et al., 1995), which is constituted from a triple repetition of a transcription activation element of the Agrobacterium tumefaciens octopine synthase gene promoter, of a transcription activation element of the mannopine synthase gene promoter, and of the mannopine synthase promoter of Agrobacterium tumefaciens, 
the rice actin promoter followed the actin intron (PAR-IRA) contained in the plasmid pAct1-F4 described by McElroy et al., (1991),
the barley HMWG (high molecular weight glutenin) promoter (Anderson O. D. et al., 1989),
the maize xcex3zein gene promoter (Pxcex3zein) contained in plasmid pxcex363 described in Reina et al., (1990) which allows for expression in the albumen of maize seed.
Accordingly, the object of this invention extends to any recombinant nucleotide sequence such as described above containing the double 35S constitutive promoter (Pd35S) of the CaMV, the promoter PCRU of the radish cruciferin gene, the promoters PGA1 or PGA6 of Arabidopsis thaliana, the chimeric promoter super-promoter PSP of Agrobacterium tumefaciens, the rice PAR-IRA promoter, the barley HMWG promoter, or the maize Pxcex3zein promoter, upstream of said cDNA or a sequence derived therefrom.
Sequences coding for an addressing peptide in the framework of the present invention may be of plant, human or animal origin.
Amongst sequences coding for an addressing peptide originating in plants, examples include:
the nucleotide sequence of 69 nucleotides (shown in the examples which follow) which codes for the 23 amino acid prepeptide (signal peptide) of sporamin A in sweat potatoes, this peptide signal allows the recombinant polypeptides of the invention to enter the secretion system of the plant cells transformed according to the invention (that is to say, principally in the endoplasmic reticulum),
the nucleotide sequence of 42 nucleotides (shown in the examples which follow) which codes for the vacuolar addressing N-terminal propeptide of 14 amino acids of sweet potato sporamin A, which allows for the accumulation of recombinant polypeptides according to the invention in the vacuoles of plant cells transformed according to the invention,
the nucleotide sequence of 111 nucleotides (shown in the examples which follow) which codes for the prepropeptide of 37 amino acids of sporamin A comprising the N-terminal part nearest the C-terminal part comprising the 23 amino acids of the aforementioned signal peptide, followed by the 14 amino acids of the aforementioned propeptide: this prepropeptide allows for entry of the recombinant polypeptides of the invention in the secretion system, and their accumulation in the vacuoles of the plant cells transformed according to the invention; the three aforementioned sequences are described in the articles of Murakami et al., 1996, and Matsuoka et al., 1991,
the barley lectin carboxy terminal propeptide, particularly as described in the articles of Schroder et al., 1993, and Bednarek et al., 1991,
and PRS (pathogenesis related protein, Corenlissen et al., 1986) which allows for secretion.
Amongst sequences which code for an addressing peptide, examples also include those coding for the peptides KDEL (SEQ. ID. NO.: 1), SEKDEL (SEQ. ID. NO.: 2) and HDEL (SEQ. ID. NO.: 3), and allowing for addressing in the endoplasmic reticulum.
The object of the invention also extends to any recombinant nucleotide sequence such as described above which contains a sequence coding for all, or part, of a vacuole addressing peptide, particularly that of sweat potato sporamin A; where this sequence codes for a vacuole addressing peptide which is located, in said recombinant nucleotide sequence, between the sequence coding for a signal peptide and that coding for said cDNA or a derived sequence thereof, in such a way that the first N-terminal amino acid of the vacuole addressing peptide is joined to the last C-terminal amino acid of the signal peptide, and wherein the last C-terminal amino acid of said addressing peptide is joined to the first N-terminal amino acid of the polypeptide coded by said cDNA or a derived sequence thereof, in the protein coded by said recombinant nucleotide sequence.
The object of the invention also extends to any nucleotide sequence such as described above which contains a sequence which codes for all or part of a vacuole addressing peptide, particularly that of barley lectin, wherein this sequence codes for a vacuole addressing peptide which is located upstream of the sequence coding for said cDNA or a derived sequence thereof, so that the first N-terminal amino acid of the vacuole addressing peptide is joined to the last C-terminal amino acid of the polypeptide coded by said cDNA or a derived sequence thereof, in the protein coded by said recombinant nucleotide sequence.